Methods of Treating Wounds With Gonyautoxins

ABSTRACT

Pharmaceutical compositions comprising tricyclic 3,4-propinoperhydropurines and uses thereof for blocking neuronal transmission which are useful in treating anal fissure and other wounds and muscle disorders are provided. Also provided are methods of treating wounds and muscle disorders by administering the composition of the invention to a muscle or in the vicinity of a muscle either topically or by injection.

FIELD OF THE INVENTION

This invention relates to pharmaceutical compositions containing heterocyclic guanidine-type compounds and uses thereof for treating wounds. More specifically, this invention relates to tricyclic 3,4-propinoperhydropurines and uses thereof for treating wounds, particularly in for use in treating anal fissure and other ailments.

BACKGROUND OF THE INVENTION

Paralytic shellfish poisoning (PSP) results from a mixture of phycotoxins that bind reversibly to a receptor site on the voltage-gated sodium channel found in excitable cells. The primary clinical symptom is an acute paralytic illness. Phycotoxins or algal toxins are produced by microscopic planktonic algae. These toxins accumulate on filter feeders such as bivalves. Consumption of phycotoxin-contaminated shellfish results in six diseases in humans: PSP, diarrhetic shellfish poisoning (DSP), amnesic shellfish poisoning (ASP), neurotoxic shellfish poisoning (NSP), ciguatera poisoning (CP) and cyanobacterial poisoning (CNP).

The phycotoxins that produce PSP have a common structure of 3,4,6-trialquil tetrahydropurine. Twenty-six naturally occurring phycotoxins have been described. These phycotoxins are non-protein, low molecular weight compounds of between 280 and 450 daltons. The GTXs are the most abundant of these phycotoxins found in shellfish extract accounting for over 80% of the total toxin content.

The high toxicity of these phycotoxins is due to reversible binding to a receptor site on the voltage-gated sodium channel on excitable cells, thus blocking the influx of sodium ions and preventing nerve and muscle cells from producing action potentials, thereby blocking neuronal transmission and causing death in mammals via respiratory arrest and cardiovascular shock. Application of small amounts of these phycotoxins can produce a flaccid paralysis of striated muscle for periods that are dose dependent.

Muscle tension may have negative effects on the healing of tissue and is one of the chief factors in determining the degree of scar formation. Surgeons have been seeking methods of reducing excessive scar formation and ways to facilitate wound healing by taking steps to overcome the effect of muscle tension on the wound healing process, including various suturing techniques, steroid injections, undermining wound edges and placing incisions in a line parallel to relaxed skin tension lines.

Anal fissure is a cut or crack in the anal canal that may extend from the mucocutaneous junction to the dentate line. This common problem causes substantial morbidity with nearly equal incidence in both sexes and is difficult to heal without intervention. Classical symptoms are pain during or after defecation that may be severe and last several hours. In many cases, bright blood may appear on the toilet paper. The cause of chronic fissure is unclear as is the reasons for complete healing. The main characteristics of this condition including the predilection for the posterior midline and the lack of granulation tissue at the fissure site are also unexplained.

Spasm of the interior anal sphincter has been associated with anal fissure. Treatment methods have focused on alleviating hypertonia of the sphincter. The most common treatment for chronic anal fissure has been lateral internal sphincterotomy. The fundamental problem with this surgical procedure is permanent changes in the control of gas, mucus and stool. Medications have been used to temporarily create the effect of sphincterotomy which reduces anal tone. Such methods include the injection of BoTox and the topical application of nitroglycerin or nifedipine ointments.

Nitroglycerine treatment of chronic anal fissure yields a healing rate of 36 to 60% within two to six months. Placebo treatment of chronic anal fissures yields a 50% healing rate. Treatment with nitroglycerine causes moderate to severe headaches and long term treatment (over three months) causes a decrease in the MRP ranging from 17 to 38% without a significant change in MVCP.

Topical application of nifedipine yields a healing rate of 50 to 94.55 within six weeks. Topical application does not produce any significant side effects and reduces MRP 11% from baseline without causing any MVCP changes. Recurrence is a frequent problem in both ointment treatments with 31% relapse occurrence in nitroglycerine treated patients having relapse and 42% relapse occurrence in nifedipine patients.

BoTox treatment is used in patients with idiopathic anal fissure with a healing rate of 60 to 96% with two to six months. The healing rate is associated with regiment dosage, which yields a 96% healing using two doses of Botox. This treatment results in reduced anal tone in all patents 5 days post-injection. Two months post-injection with BoTox MRP was reduced 28.4% without any significant change in MVCP. BoTox treatment produces a chemical denervation that lasts over 3 months along with a 10% temporal fecal incontinence and a 20% relapse occurrence.

There remains a need for a safe, effective and potentially long-lasting treatment for wound healing and/or to reduce scar tissue formation.

SUMMARY OF THE INVENTION

In accordance with the objects of the invention, novel methods for the use of paralytic shellfish poisons are provided.

In one aspect of the invention, methods of treating wounds with compositions of the invention including one or more tricyclic 3,4-propinoperhydropurines are provided.

In another aspect of the invention, methods for treating anal fissure are provided.

In another aspect of the invention, the methods of the invention compositions of the invention are used in combination with an effective amount of another neurotoxin and/or a local anesthetic.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, it has been found that compositions comprising heterocyclic guanidine-type compounds, and more specifically tricyclic 3,4-propinoperhydropurines, can be used for certain clinical applications, to avoid or minimize the need for surgery, or to avoid or minimize side effects, allergies, immune rejection or hematoma. In some aspects, the invention is based, in part, on the reduction or elimination of the tension acting on a wound by a muscle. In some aspects, the invention is based, in part, on the surprising, long lasting effects that are provided by certain heterocyclic guanidine-type compounds in the various methods of the invention.

In some aspects, the present invention relies on administration of a chemodenervating agent to relax or paralyze muscles capable of exerting tension thereby providing improved healing of a wound or treatment of a muscle-related disorder. Other advantages, such as a surprising, long lasting effect, and the ability to avoid talking other measures to reduce muscle tension, are realized by certain aspects of the present invention.

Before describing the present invention in detail, it is to be understood that this invention is not limited to specific drug delivery systems, device structures, enhancers or carriers. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

It must be noted that, as used in this specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a pharmacologically active agent” includes a mixture of two or more active agents, reference to “an enhancer” includes mixtures of two or more enhancers, and the like.

In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set out below.

The terms “treating” and “treatment” as used herein refer to reduction in severity and/or frequency of symptoms, elimination of symptoms and/or underlying cause, prevention of the occurrence of symptoms and/or their underlying cause, and improvement or remediation of damage. The present method of “treating” a patient, as the term is used herein, thus encompasses both prevention of one or more symptoms or underlying causes in a predisposed individual, as well as treatment of one or more symptoms or underlying causes in a clinically symptomatic individual.

The terms “active,” “active agent,” “drug” and “pharmacologically active agent” are used interchangeably herein to refer to a chemical material or compound that induces a desired effect, and include agents that are therapeutically effective, prophylactically effective, or cosmetically effective. Also included are derivatives, metabolites and analogs of those compounds or classes of compounds specifically mentioned which also induce the desired effect.

By “therapeutically effective” amount is meant a nontoxic but sufficient amount of an active agent to provide the desired therapeutic effect.

By “transdermal” drug delivery is meant administration of a drug to the skin surface of an individual so that the drug passes through the skin tissue and into the individual's blood stream, thereby producing a systemic effect. The term “transdermal” is intended to include “transmucosal” drug administration, i.e., administration of a drug to the mucosal (e.g., sublingual, buccal, vaginal, rectal) surface of an individual so that the drug passes through the mucosal tissue and into the individual's blood stream.

The term “topical administration” is used in its conventional sense to mean delivery of a topical drug of a pharmacologically active agent to the skin or mucosa, as in, for example, the treatment of various skin disorders. Topical drug administration, in contrast to transdermal administration, provides a local rather than a systemic effect. Unless otherwise stated or implied, the terms “topical drug administration” and “transdermal drug administration” are used interchangeably.

As used herein, “an effective amount” is that amount sufficient to interfere with neuronal transmission by blocking at least some of the presynaptic release of the neurotransmitter acetylcholine in the neuromuscular plate, thus interfering with transmission, paralyzing the muscle and preventing it from contracting, or producing a relaxation of contracted muscles.

Amounts are given in units of activity. One unit of activity corresponds to an amount of the composition of the invention necessary to block the muscular contractions of the crural biceps of a 20 gram CF1 albino or a BALB-C strain mouse leg for 1.5 to 2.0 hours. The toxin is intramuscularly injected in the crural biceps of the mouse right leg in a volume of 0.5 ml. The left leg is used as a control.

In order to measure the amount of toxin used in each dose, High Performance Liquid Chromotography (HPLC) analysis can be performed with on line fluorescence detection (HPLC-FLD). This method allows the measurement of the mass of each toxin in any mixture, extract or pharmaceutical formulation.

“Carriers” or “vehicles” as used herein refer to carrier materials suitable for transdermal drug administration. Carriers and vehicles useful herein include any such materials known in the art, which is nontoxic and does not interact with other components of the composition in a deleterious manner.

The term “aqueous” refers to a formulation or drug delivery system that contains water or that becomes water-containing following application to the skin or mucosal tissue.

The methods of the invention may be used for wound healing. More specifically, the methods of the invention are useful for the treatment of conditions including, but not limited to, improving wound healing, achalasia and anal fissure.

The compositions of the invention comprise an effective amount of at least one tricyclic 3,4-propinoperhydropurine (represented by formula (I) set forth below:

wherein R₁ and R₅ are independently selected from the group consisting of —H and —OH; R₂ and R₃ are independently selected from the group consisting of —H and —SO₃; and R₄ is selected from the group consisting of —H, —OH, —COONH₂, —COONHSO⁻ ₃ and —COOCH₃, with the proviso that either one of R₂ and R₃ must be —OSO⁻ ₃, or R₄ must be —COONHSO⁻ ₃, and a pharmacologically acceptable topical carrier.

Preferred tricyclic 3,4-propinoperhydropurines in accordance with the present invention are the gonyautoxins (hereinafter “GTX”) of the formula (I) as set forth in the table below. Compound R₁ R₂ R₃ R₄ R₅ Gonyautoxin 1 —OH —H —OSO⁻ ₃ —COONH₂ —OH Gonyautoxin 2 —H —H —OSO⁻ ₃ —COONH₂ —OH Gonyautoxin 3 —H —OSO⁻ ₃ —H —COONH₂ —OH Gonyautoxin 4 —OH —OSO⁻ ₃ —H —COONH₂ —OH Gonyautoxin 5 —H —H —H —COONHSO⁻ ₃ —OH

In one aspect of the invention, the pharmaceutical compositions of the invention comprise at least one GTX compound selected from GTX 1, GTX 2, GTX 3, GTX 4 and GTX 5. In other aspects of the invention, the pharmaceutical compositions comprise a mixture of tricyclic 3,4-propinoperhydropurines. For example, mixtures of two or more GTX compounds are contemplated. One embodiment of the invention employs a mixture of GTX 2 and GTX 3, in approximately a 2:1 ratio, respectively. Another embodiment of the invention employs a mixture of GTX 1, GTX 2, GTX 3 and GTX 4. It should be understood by those of skill in the art that, subject to the conditions set forth with respect to the formula (I) above, other mixtures and combinations of tricyclic 3,4-propinoperhydropurines are within the scope of this invention.

Unlike Botulin A toxin, these preparations are stable at room temperature, do not require refrigeration, are sterilizable, are substantially non-allergenic, are not peptide in nature, act substantially immediately, and may be applied repeatedly without significant, adverse side effects.

The compounds of the formula (I) may be purified from dinoflagellates or cyanobacterias, and also may be accumulated by highly-contaminated mollusks, from which they may also be purified. Any pharmacologically acceptable carrier may be employed, including, but not limited to, water, acetic acid, and saline solutions. Acetic acid and 0.09% sodium chloride solution are preferred carrier materials.

Alternatively, the pharmaceutical compositions of the invention include at least GTX compound, as discussed above, and additionally comprise at least one compound selected from the group consisting of saxitoxin (STX), neosaxitoxin, decarbamoylsaxitoxin, tetanus toxin, and Botulin A toxin. The combination may be used in any of the applications in which the compositions of the invention are used.

In another aspect, the compositions of the invention are used in combination with an effective amount of a local anesthetic. In this aspect, the pharmaceutical compositions of the invention comprise an effective amount of a local anesthetic such as enzocaine, tetracaine, mepivacaine, prilocaine, etidocaine, bupivacaine, lidocaine or similar local anesthetics and combinations thereof and an effective amount of at least one tricyclic 3,4-propinoperhydropurine. Compositions of the invention may also be used in combination with one or more neurotoxins and one or more local anesthetics, if desired.

Without being bound by theory, when applied locally, these compounds appear to effect the antispasmodic action by blocking the spreading of nervous impulse, or neuronal transmission, by reversibly binding to the sole biological molecular receptor, i.e. the voltage gated sodium channel, present in all neurons and excitable cells. By binding to this channel, there is no entry of sodium to the neuronal cell; depolarization does not occur and, therefore, propagation of the impulse is stopped. This action mechanism blocks at least some of the presynaptic release of the neurotransmitter acetylcholine in the neuromuscular plate, thus interfering with neuromuscular transmission, paralyzing the muscle and preventing it from contracting, or producing a relaxation of muscles contracted by pathological problems.

The pharmaceutical preparations of the invention may be applied in any suitable manner, such as topically, transdermally or by injection. In the preferred embodiment, the preparations of the invention are applied locally in the vicinity of the muscle that is to be paralyzed or prevented from contracting. The application should be in amounts sufficient to provide from 1-1000 units of activity to the muscle, per dose, more preferably, from 10-1000 units of activity, per dose. The effect is immediately apparent, generally occurring within a maximum of 30 seconds to five minutes after penetration of the active compound through the skin. The maximum effect is generally achieved within 15 minutes of penetration of the active compound through the skin. Its effective duration depends on the dose administered, the muscle in question, as well as the volume and specific composition administered. This is the pattern for all clinical applications and pathologies.

It has been surprisingly found that certain methods of the present invention not only provide practically immediate anesthetic and muscle relaxant effects, but also provide beneficial muscle relaxation effects over an unexpectedly long period. As a result, certain methods of the present invention are particularly well-suited to wound healing and treatment of the other indications mentioned above, since the unexpected longer term muscle relaxation effects have been found to facilitate the healing process.

More preferably, each dose includes more than 32 units of activity up to a maximum of about 5000 units of activity. Even more preferably, each dose includes more than 32 to about 1000 units of activity, more preferably, from more than 40 to about 1000 units of activity, and even more preferably, from about 50 to about 500 units of activity. Most preferably, each dose includes about 75 to about 200 units of activity. Doses may be repeated from time-to-time, as needed, to continue muscle relaxation until the desired effect is obtained.

According one method of the present invention, mammals are treated by direct, local injection of a composition of the invention into a muscle or in the vicinity of a muscle. In some embodiments, the muscle at the location of injection exhibits elevated tone or spasms, though this is not necessary for the effectiveness of all treatments.

Each injection may be limited to not more than two milliliters of composition, including solvents, adjuvants and/or carrier materials. Administration may be accomplished, for example, using a one milliliter, tuberculin-type disposable syringe with a twenty-seven to thirty gauge needle. Alternatively, for certain methods, it may be desirable to administer the dosage using an endoscope such as that described in U.S. Pat. No. 5,674,205, the disclosure of which is hereby incorporated by reference for the purpose of providing details of an endoscope suitable for use in the method of the present invention.

Alternatively, the pharmaceutical compositions of the invention may be applied locally in the form of a topical preparation. To form a topical preparation, an effective amount of the pharmaceutical composition of the invention is added to a pharmacologically acceptable topical carrier. The composition, when applied topically, may be in any form suitable for application to the body surface, and may comprise, for example, a cream, lotion, solution, gel, ointment, paste or the like, and/or may be prepared so as to contain liposomes, micelles, and/or microspheres. The topical preparation may include from about 0.0001% to about 0.01% by weight of the GTX compounds, based on the weight of the preparation. Alternatively, the topical preparation may include from about 0.001% to about 0.01% by weight of the GTX compounds, based on the weight of the preparation.

The composition may be directly applied to the body surface or may involve use of a drug delivery device. Thus, a formulation or drug reservoir may be aqueous, i.e., contain water, or may be nonaqueous and used in combination with an occlusive overlayer so that moisture evaporating from the body surface is maintained within the formulation or transdermal system during drug administration. In some cases, however, e.g., with an occlusive gel, a non-aqueous formulation may be used with or without an occlusive layer.

In the following examples, “dose” refers to the amount of toxin used during one administration to a patient. A dose may be applied to one or more sites. “Treatment” refers to the total number of doses given to a patient to achieve the desired effect. 100 units of the mixture of GTX 2 and GTX 3, determined by mouse assay, is equivalent to 25 μg of GTX 2 and GTX 3, as determined by HPLC-FLD.

EXAMPLE 1

Ten healthy male adults between 24 and 48 years old with normal sphincter tone (pressure at rest less than 72 mmHG as determined by anorectal manometry) were studied. The test subjects had no anorectal pathologies like hemorrhoids, fistula or abscesses. Before intervention, tests were performed including anorectal manometry, electromyography, a hemogram, a basic metabolic panel and a urinalysis.

A dosage of 100 units of activity of a combination of GTX-2 and GTX-3, in approximately a 2:1 ratio, in a total volume of 1.0 ml was locally infiltrated into both sides of the anal internal sphincter (0.5 ml each side) with an insulin syringe (25 gauge). The mixture of GTX-2 and GTX-3 was purified from shellfish collected in the Chilean Patagonia fjord that was found to be highly-contaminated with PSP toxins using standard purification methods.

Two minutes post injection an anorectal manometry was performed. Resting and voluntary contraction pressures were measured and recorded and compared to the baseline measurements. Recto-anal and cortical-anal reflexes were also measured and recorded in each participant before and after toxin injection. The anal canal pressure was recorded by a stationary pull-through technique with a water-filled micro-balloon and external transducer (PVB) perfusion equipment. Recording and analysis of the tracing were made by a computerized system (8 channels polygraph ID, Metronic Polygraph with Polygram 98 ver. 2.2).

Anal resting pressures were recorded in millimeters of mercury with the stationary pull-through technique and the computer identified the mean pressure. The maximal voluntary contraction was assessed by evaluation of the voluntary contractions of the anal sphincter in each participant. Amplitude was expressed in milliliters of mercury.

The participants were evaluated at 24 and 48 hours, after one week and after 12 days. Blood and urine samples were collected from each participant a week after the injection for laboratory analysis. Toxin levels in the urine were measured 4 hours after injection. Pulse and blood pressure, as well as any side effects and pain scores were recorded at each visit. The injection pain scores and the pain two minutes after the injections were determined by consulting the participants using a 1-10 pain scale, with 10 being the maximum value of pain.

Long-term outcomes were determined after a median follow up of 12 months via personal communication with the participants and by clinical examination at the request of the participants.

No participant dropped out of the study and none had any adverse or negative side effects. Laboratory tests did not show any significant changes and no toxin was detectible in the urine samples collected 4 hours after injection. Toxin detection was performed by analytical high performance liquid chromatography (HPLC) with on line fluorescent detection; the detection limit of this method is 1 microgram in 10 ml. This also corresponds to the fact that PSP toxins, once injected, immediately enter the extracellular medium producing a dilution.

Table 1 is a summary of the test results. As shown in Table 1, all participants showed anal sphincter relaxation within 30 seconds after injection, as determined by clinical anorectal examination during the injection and then after two minutes using anorectal manometry. The participants all stated that they felt the anal anesthesia for an average time of 59.5 minutes±7.12 minutes (mean value±Stand. Dev.) and sphincter hypotonical sensation for 40.0±4.20 minutes. None of the participants showed flatus incontinence or transitory fecal incontinence. TABLE 1 Symptoms and Side Effects Healthy Voluntary Adults 10 Pain During Injection (On a scale of 1- 5.55 ± 0.4 10) Pain 2 Minutes Post Injection 100% without pain Anal Anesthetic Sensation (time) 59.50 ± 7.12 minutes (n = 9) Sphincter Relaxation Sensation 40.0 ± 4.20 minutes (n = 9) Flatus Incontinence None Fecal Incontinence None Clinical Evaluation Immediate Relaxation Side Effects None Other All asymptomatic after 24 hours

Table 2 is a summary of the anorectal manometric recordings. Manometric recording showed a significant decrease in the anal maximal voluntary contraction pressure. Two minutes after injection the pressure was 55.2±6.2% (mean value±standard dev.) of the base line values, a diminishing of 44.8%. Twenty-four hours after injection, the drop in the anal maximal voluntary contraction pressure increased to 53% of the baseline value. Fifteen days after injection all of the anal maximal voluntary contractions had returned to the base line values. Electromyography (EMG) recorded before the injection and after the injection indicated that the injection nearly eliminated muscle activity. At the 12-month follow up examination (long term follow-up) none of the participants showed any adverse or systemic side effects (i.e. anorectal problems). TABLE 2 Anorectal Manometry Recordings Maximum Resting Pressures 66 ± 5.8 mm Hg (n = 10) Maximum Voluntary Contraction Pressure (MVCP) Pre-Injection 126 ± 11.5 mm Hg (n = 10) 2 minutes Post-Injection 69.5 ± 5.8 mm Hg (n = 10) 24 hours Post Injection 59.2 ± 7.2 mm Hg (n = 5) % MVCP 2 Minutes Post-Injection 55.2 ± 6.2% (n = 9) % MVCP 24 Hours Post-Injection 47.0 ± 6.8% (n = 5) Recto-Anal Reflex (after 60 cc) 100% Retention Cortical-Anal Reflex 100% Retention

As can be seen from Table 2, complete anal sphincter relaxation after administration of the mixture of GTX-2 and GTX-3 was achieved in 100% of the participants immediately. This indicates that the injection of these toxins locally produces paresis in the sphincter. This relaxation was confirmed by anal clinical evaluation as performed by coloproctologists during the injection. The injection of the toxin did not produce any long lasting side effects such as flatus or fecal incontinences and the recto-anal and cortical-anal reflexes remained normal. The toxin level injected blocks extra contraction of the muscle but leaves sufficient strength to provide for normal performance.

The local peripheral application of toxins interferes with neuromuscular transmission, altering the action potential which results in a temporarily paralysis of the sphincter. The mode of action of GTX is directed to the injected muscle leaving other muscles in the area unaffected. The paretic effects indicate that local injection of the sphincter lasts for 12 days. As there are no side effects, the use of this toxin injected locally is safe and effective particularly for use in the anal sphincter. The immobilization of healing tissue is a fundamental therapeutic principle and treatments using paralytic toxins may be effective in the remission of other pathologies such as blepharospasm, tics, tremors, bruxism, hemifacial spasm, cervical dystonia, cerebral palsy, muscle spasm pain such as writer or musician cramps, hand tremors, spasmodic dystonia to list just a few conditions of muscle hypertonicity causing stiff and awkward movements.

EXAMPLE 2

Fifty adults between the ages of 18 and 70 with symptomatic anal fissures were evaluated for inclusion in a study to test the effects of GTX local infiltration efficacy in the treatment of anal fissure via the reduction of anal tone to promote the healing process and eliminate the need for surgical intervention as well as provide a safe and effective alternative to ointment treatments.

Patients were evaluated for the following inclusion criteria: evidence of anterior, posterior or both circumscribed ulcers, induration at the edges and posterior of horizontal fibers of the interior anal sphincter, with symptoms of post defecatory or permanent pain, bleeding or both. Patients with an anal fissure history of two or month duration were considered to be chronic. Table 3 contains a detail of the patients selected for study. Patients younger than 18 or older than 70 years of age were excluded from this study as were those patients that were pregnant or had anal fissure associated with other conditions such as hemorrhoids, fistula or anal abscess. TABLE 3 Anal Fissure Patient Characteristics Characteristic Patients (N = 50) Sex (M/F) 17/33 Mean Age (Years) 35.9 ± 13.1 Spontaneous Pain (No. of Patients) 46 Post-defecatory Pain (No. of Patients) 50 Bleeding (No. of Patients) 49 Mean Duration of Symptoms (Months) 24.4 ± 39.7 Acute/Chronic 17/33 Localization (Posterior/Anterior/Bilateral) 32/8/5 History of Constipation (No. of Patients) 42 Previous Conservative Treatment (No.) 36 Previous Surgical Treatment (No.)  2

The healing criteria and primary end points of the study were pain relief and fissure epithelization. The participants were fully informed about the action and mechanism of GTX and all included patients supplied their written consent. The study was a randomized double-blind trial in which the patients were diagnosed and injected with a mixture of GTX-2 and GTX-3 or placebo solution of 0.9% sodium chloride. The study was opened for humanitarian and ethical reasons after 15 placebo treated patients showed either no improvement or a worsening of their conditions and patients injected with GTX showed marked improvement after three weeks treatment.

The test dosage consisted of 100 units of activity of a mixture of GTX-2 and GTX-3 in 1.0 ml volume, which was locally infiltrated into both sides of the anal fissure, in the internal anal sphincter, in equal volumes. The injection was accomplished with an insulin needle (25 gauge).

Anal pressures were measured and recorded as described above. Patents were clinically evaluated after 7, 14 and 28 days post-injection. Pain during injection and two minutes were evaluated by asking the patients to rate their pain on a scale of 1 to 10, with 10 being the maximum pain value. No stool softeners, bulk laxatives or sitz baths were prescribed. Long-term outcomes where determined at the 12 month follow-up examination. TABLE 4 Symptoms and Side-Effects in Patients After Treatment Number of Doses Needed 2.6 ± 1.3 Mean Healing Time (Days) 17.6 ± 9.0  Pain During Injection 5.8 ± 2.3 Pain 2 minutes Post Injection 1.6 ± 1.2 Duration of Bleeding (Post 1^(st) Injection, 1.2 ± 0.9 Days) Duration of Pain (Post 1^(st) Injection, Days) 2.1 ± 0.7 Flatus Incontinence None Fecal Incontinence None Digital Exam (Immed. Sphincter Relax) 100% Side Effects None in 100% of Patients

TABLE 5 Anorectal Manometry Recordings Maximum Resting Pressures (MRP) (N = 50 patients) Before Injection 108.8 ± 24.9 2 Minutes Post Injection  61.1 ± 22.8 % MRP 2 Minutes Post Injection  56.2 ± 12.5 Maximum Voluntary Contraction Pressures (MVCP) (N = 50 patients) Pre-Injection (mmHg) 159.9 ± 32.8 2 minutes Post Injection 118.8 ± 12.7 % of MVCP 2 minutes Post Injection  74.3 ± 13.1 Reflexes Recto-Anal (% Maintained) 100 Cortical (% Maintained) 100

None of the fifty patients enrolled in the study had any adverse or negative side effects during or after the trial. As shown in Table 5, 100% of the patients showed immediate post-injection anal sphincter relaxation as detected by digital examination and anorectal manometry. During the digital examination, coloproctologists detected the anal tone reduction and all the patents declared that they felt anal anesthesia after injection. The seven day post-injection examination showed greatly reduced anal tone in all patients. No incontinence was observed and all the patients maintained the recto-anal and cortical-anal reflexes (see Table 4).

Table 5 demonstrates a significant decrease in the maximum resting pressures (MRP) in all the patients as indicted by the manometric recordings. The MRP recordings fell from 108.8±24.9 to 61.1±22.8 mmHg (Mean value±Standard Deviation) decreasing to 56.2±12.5 of MRP baseline values. This represents a mean reduction of 43.8%. Similarly, maximum voluntary contraction pressure (MVCP) decreases to 74.3±13.1 of MVCP baseline values, a 25.7% mean reduction.

Patients with acute anal fissures stopped bleeding within 48 hours of injection. By the day seven post-injection examination, these patients showed epithelization of the lesion with modest pain only after defecation. At the day 14 post-injection examination, the patients all showed totally epithelization with scar formation.

Patients with chronic anal fissures also stopped bleeding within 48 hours. By the day seven post-injection digital examination, 50% of the patients with chronic anal fissures showed epithelization similar to the acute patients. Nevertheless, epithelization was evident by the 14-day post-injection examination and epithelization was complete by the 28-day post-injection examination in 98% of the patients with chronic anal fissure. All patients were post-defecation pain free and asymptomatic.

One patient relapsed 3 months post-injection and required surgical intervention. This patient was a 59 year old female with an eight year history of recurrent anal fissures accompanied with periods of intense pain and bleeding, which was further complicated with sub-fissural infiltration and bleeding tendency. Her anaorectal manometry reading at baseline showed an MRP of 118 mmHg and a MVCP of 171 mmHg. The remaining patients in the study remained asymptomatic and healthy. After a 38 month follow-up, only two patients had presented a recurrence of the anal fissure requiring a different treatment.

The results indicate that 100% of the patients experienced relaxed anal sphincter immediately after infiltration. Paresis is produced in the internal sphincters and a concurrent reduction of anal tone is provided. Neither flatus or fecal incontinences were observed and all patients maintained functional anal and cortical-anal reflexes suggesting that infiltration blocks extra contraction of the muscle but leaves sufficient strength for normal muscle performance. Repeated injections did not produce any problems or side effects. The paretic effect lasts for more than a week. At the 12-month examination, no side effects were observed.

Of the fifty patients in the study, 3 of the acute and 33 of the chronic patients had been previously conservatively treated with sitz baths, stool softeners, high fiber diets, bowel regulation and topical ointments to no effect (not cured). The healing rate of this study was 98% within 28 days for all patients, but the 17 acute patients were healed within 15 days. These results show a higher efficacy than other pharmaceutical treatments (BoTox, nitroglycerine or nifedipine).

This example emphasizes that tissue immobilization is a fundamental therapeutic principle of healing. Treatment with toxins creates a chemical denervation which produces muscle paralysis when toxins are injected locally. As only 20 days are required to heal anal fissures, the production of three or more months of chemical denervation appears to be unnecessary. GTX treatment reduces the chemical denervation induction time period. GTX, when given in a 100 units of activity dosage, yields a reversible chemical denervation of twelve days duration, in contrast to BoTox treatment which produces an irreversible chemical denervation of three months or more duration along with proteolytic structural damage. Muscle inactivation persists until new fibrils grow from the nerve forming new junction plates at new areas on the muscle cell wall. TABLE 6 Comparison of published results on the treatment of patients with chronic anal fissure using ointments, botulinum toxins and PSP toxins Healing rate at 3 Temporary # of months incontinence Recurrence Complications Patients Dose (%) (%) (%) (%) References OINTMENTS + CONSERVATIVE TREATMENTS 26 Nitroglycerin 36 31 Moderate Dis Colon Rectum to severe 2002; 46: 805-8 headaches 25 Nitroglycerin 60 12 Headaches N Eng J Med 1999; Healing rate 341: 65-9 at 6 weeks (%) 141 Nifedipine 50 5 Dis Colon Rectum 1999; 42: 1011-15 55 Nifedipine 94.5 5.4 Dis Colon Rectum 2002; 45: 1468-75 SURGERY 250 100 1 Dis Colon Rectum 1995; 38: 378-82 696 100 24 (32 gases) 13 Colo-Proctology 1987; 9: 49-53 829 100 14 (26 gases) Br J Surg 1989; 76: 431-4 TOXINS Hetaling rate at Units 6 weeks (%) 10 15 BOTOX 70 10 20 10 Lancet 1994; 344: 1127-28 12 5 BOTOX 83 0 8 0 Dis Colon Rectum 1994; 37: 1321-4 54 5 BOTOX 78 6 6 11 Dis Colon Rectum 1995; 38: 781 5 Nr DYSPORT 60 0 0 0 82 100 5 BOTOX 82 7 6 0 Dis Colon Rectum 1997; 40: 1029-32 36 10/15 BOTOX 65/81 0 0 0 83 25 20 BOTOX 88 4 0 0 N Engl J Med 1998; 338: 217-20 57 15/20 BOTOX 44/68 0 0 0 Ann Surg 1998; 228: 664-9 69 10-21 BOTOX 48-70 0 37-52 0 Dis Colon Rectum 1999; 42: 1016-21 50 20/40 76/80 4/12 4/8 0 Dig Dis Sci DYSPORT 199; 44: 1588-9 25 20 BOTOX 96 (2 0 0 0 N Engl J Med doses, 6 1999; 341: 65-69 months) 50 20 BOTOX 74 0 0 0 Am J Surg Healing rate 2000; 179: 46-50 at 3 weeks 82 100 PSP 98.8 (1 en 0 0 0 toxins 82)

Thus, as Table 6 demonstrates, GTX provides a safer treatment regiment for healing acute and chronic anal fissures than the current therapeutic methods such as BoTox or surgery. GTX treatment does not produce the inconveniences of flatus or fecal incontinence, nor does it require 5 to 7 weeks healing time. GTX treatment does not produce permanent sphincter damage resulting in long term hospitalization.

EXAMPLE 3

One unit of activity corresponds to an amount of the composition of the invention necessary to block the muscular contractions of the crural biceps of a 20 gram CF1 albino strain mouse leg for 1.5 to 2.0 hours. The toxin was intramuscularly injected in the crural biceps of the mouse right leg in a volume of 0.5 ml. The left leg is used as a control. This was done in three mice and the paralyzing effect was tested every 30 minutes for the first two hours, and then every 2, 4, 8 hours and overnight. Depending on the dose injected, the paralyzing effect can last 24 hours or longer. This example confirms the reversible nature of the effect of the toxins of the present invention and demonstrates that the duration of the effect can be controlled by varying the dosage of the toxins.

EXAMPLE 4

Doses of 200 units up to 5,000 units of a mixture of GTX 2 and GTX 3, as employed in example 2, have been administered by injection in the internal anal sphincter, in normal volunteers. These doses were well tolerated, without adverse or negative side effects. The volunteers remained healthy during and after the local injection of this relatively large amount of toxins.

EXAMPLE 5 Ointment for Treatment of Anal Fissure

Petrolatum 75.000% w/w Sorbitan sesquioleate 10.000% w/w White Wax 10.000% w/w Toxins GTX2/GTX3 0.01-0.0001% w/w Water to 100%. Purpose of Ingredients: Petrolatum: Emollient ointment base Sorbitan sesquioleate: Emulsifier, penetration enhancer White Wax: Thickener, Stabilizer Compounding Procedure:

Melt the petrolatum, sorbitan sesquioleate and white wax at 60 degrees C. and mix until uniform. Slowly incorporate aqueous solution of toxin and continue mixing until ointment congeals.

Twenty patients with anal fissures were entered into a study which had the same protocol as that described above in Example 2, except that a different formulation and mode of administration of toxin were employed. In this case, an ointment containing from 10 to 200 Units of a mixture of GTX2/GTX3 was applied to the area of the anal sphincter around the muscle tissue including the painful area where the anal fissure is exposed. A physician, with a 10-minute massage, did this topical application.

Ano-rectal manometry was performed before and 10 minutes after the application of the ointment. The pressure measurements showed no significant change after the application of the cream. Patients were sent home with the cream and instructions to apply the cream twice a day (morning and night, about every 12 hours). The patients were followed up via weekly visits to the physician. The patients started to present re-epithelialization of the fissures after the first, second and third week, and the healing was completed in between the third to the fifth week of treatment, depending of the type of anal fissure (acute or chronic or posterior, anterior, both).

The foregoing detailed description of the invention has been presented for the purpose of illustration and description only and is not to be construed as limiting the invention. The scope of the invention is to be determined from the claims appended hereto. 

1. A method of facilitating the healing of a wound comprising the step of administering to a patient in need of such treatment an effective amount of a composition comprising at least one tricyclic 3,4-propinoperhydropurine represented by formula (I) set forth below:

wherein R₁ and R₅ are independently selected from the group consisting of —H and —OH; R₂ and R₃ are independently selected from the group consisting of —H and —SO₃; and R₄ is selected from the group consisting of —H, —OH, —COONH₂, —COONHSO⁻ ₃ and —COOCH₃, with the proviso that either one of R₂ and R₃ must be —OSO⁻ ₃, or R₄ must be —COONHSO⁻ ₃, and a pharmacologically acceptable carrier.
 2. The method of claim 1, wherein the composition is injected into a muscle.
 3. The method of claim 1, where in the method is applied to a patient afflicted with anal fissure.
 4. The method of claim 1, wherein the at least one compound of the formula (I) is selected from the group consisting of: GTX-1, GTX-2, GTX-3, GTX-4 and GTX-5.
 5. The method of claim 1, wherein the composition comprises both GTX-2 and GTX-3.
 6. The method of claim 1, wherein the effective amount of said composition contains from about 1 to about 1000 units of activity.
 7. The method of claim 1, wherein the effective amount of said composition contains from more than 32 to about 1000 units of activity.
 8. The method of claim 1, wherein the effective amount of said composition contains from about 50 to about 1000 units of activity.
 9. The method of claim 1, wherein the effective amount of said composition contains from about 50 to about 500 units of activity.
 10. The method of claim 1, wherein the effective amount of said composition contains from about 75 to about 200 units of activity.
 11. The method of claim 1, wherein said composition further comprises a neurotoxin selected from the group consisting of saxitoxin, neosaxitoxin, decarbamoylsaxitoxin, tetanus toxin, and Botulin A toxin.
 12. The method of claim 1, wherein said composition further comprises a local anesthetic.
 13. The method of claim 12, wherein the local anesthetic is selected from the group consisting of: benzocaine, tetracaine, mepivacaine, prilocaine, etidocaine, bupivacaine, lidocaine.
 14. The method of claim 1, wherein the composition is injected into the internal anal sphincter.
 15. The method of claim 1, wherein the method is applied to a patient afflicted with at least one ailment selected from the group consisting of achalasia, and anal fissure.
 16. The method of claim 1, wherein the method is applied to a patient with a wound for the purpose of facilitating the healing of the wound.
 17. The method of claim 15, wherein the effective amount of said composition contains from about 50 to about 1000 units of activity.
 18. The method of claim 16, wherein the effective amount of said composition contains from about 50 to about 1000 units of activity.
 19. The method of claim 1, wherein the composition is applied topically.
 20. The method of claim 19, wherein the topical composition comprises from about 0.0001% to about 0.01% by weight of one or more compounds of the formula I, based on the total weight of the composition. 